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Structure of the inhibitor W7 bound to the regulatory domain of cardiac troponin C.

Hoffman RM, Sykes BD - Biochemistry (2009)

Bottom Line: The protein-W7 interface is defined through a three-dimensional {(1)H,(13)C}-edited-{(1)H,(12)C}-detected NOESY NMR experiment, and other aspects of the structure are modeled as perturbations to previously known coordinates and restraints.The structure supports the previously proposed troponin I blocking mechanism for the activity of W7 in striated muscle and suggests a role for the flexible tail of W7 in stabilization of the bound state.This clarifies the structure-activity relationships of W7 and implicates an electrostatically mediated component of activity in common analogues of W7, including the antipsychotic trifluoroperazine and the cardiotonic levosimendan.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

ABSTRACT
The calmodulin antagonist W7 binds to troponin C in the presence of Ca(2+) and inhibits striated muscle contraction. This study integrates multiple data into the structure of the regulatory domain of human cardiac troponin C (cNTnC) bound to Ca(2+) and W7. The protein-W7 interface is defined through a three-dimensional {(1)H,(13)C}-edited-{(1)H,(12)C}-detected NOESY NMR experiment, and other aspects of the structure are modeled as perturbations to previously known coordinates and restraints. The structure determination protocol optimizes the protein-W7 contacts prior to the introduction of protein-W7 steric interactions or conformational changes in the protein. The structure determination protocol gives families of conformers that all have an optimal docking as assessed by satisfaction of the target function. The structure supports the previously proposed troponin I blocking mechanism for the activity of W7 in striated muscle and suggests a role for the flexible tail of W7 in stabilization of the bound state. This clarifies the structure-activity relationships of W7 and implicates an electrostatically mediated component of activity in common analogues of W7, including the antipsychotic trifluoroperazine and the cardiotonic levosimendan.

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Related in: MedlinePlus

Simulation of cNTnC·Ca2+−W7 binding equilibria. The downfield W7 signal (H8) from titration 2 (Figure 3) can be fit to an n + 1 site, sequential binding model, where n is the number of secondary (nonspecific) binding sites. The circles are the data points, and the closely spaced plus signs reflect the numerical simulation. The parameter values used are as follows: Kd1 = 100 μM, Kd2 = 0.43 pM, and n = 4.
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fig4: Simulation of cNTnC·Ca2+−W7 binding equilibria. The downfield W7 signal (H8) from titration 2 (Figure 3) can be fit to an n + 1 site, sequential binding model, where n is the number of secondary (nonspecific) binding sites. The circles are the data points, and the closely spaced plus signs reflect the numerical simulation. The parameter values used are as follows: Kd1 = 100 μM, Kd2 = 0.43 pM, and n = 4.

Mentions: Binding of cNTnC·Ca2+ to W7, monitored with 1D 1H spectra. A buffered stock solution of cNTnC·Ca2+ was used, affording more control over the pH changes over the titration. The protein:W7 ratio is shown to the right of each spectrum. cNTnC·Ca2+ binding induces chemical shift changes. The binding curve rises more steeply than would be observed for 1:1 binding. The equilibrium can be modeled in terms of a microscopic affinity similar in magnitude, however (Figure 4). Assignments for W7 in the unbound state (Figures 1 and 2) were transferred to those of the bound state through these spectra.


Structure of the inhibitor W7 bound to the regulatory domain of cardiac troponin C.

Hoffman RM, Sykes BD - Biochemistry (2009)

Simulation of cNTnC·Ca2+−W7 binding equilibria. The downfield W7 signal (H8) from titration 2 (Figure 3) can be fit to an n + 1 site, sequential binding model, where n is the number of secondary (nonspecific) binding sites. The circles are the data points, and the closely spaced plus signs reflect the numerical simulation. The parameter values used are as follows: Kd1 = 100 μM, Kd2 = 0.43 pM, and n = 4.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697600&req=5

fig4: Simulation of cNTnC·Ca2+−W7 binding equilibria. The downfield W7 signal (H8) from titration 2 (Figure 3) can be fit to an n + 1 site, sequential binding model, where n is the number of secondary (nonspecific) binding sites. The circles are the data points, and the closely spaced plus signs reflect the numerical simulation. The parameter values used are as follows: Kd1 = 100 μM, Kd2 = 0.43 pM, and n = 4.
Mentions: Binding of cNTnC·Ca2+ to W7, monitored with 1D 1H spectra. A buffered stock solution of cNTnC·Ca2+ was used, affording more control over the pH changes over the titration. The protein:W7 ratio is shown to the right of each spectrum. cNTnC·Ca2+ binding induces chemical shift changes. The binding curve rises more steeply than would be observed for 1:1 binding. The equilibrium can be modeled in terms of a microscopic affinity similar in magnitude, however (Figure 4). Assignments for W7 in the unbound state (Figures 1 and 2) were transferred to those of the bound state through these spectra.

Bottom Line: The protein-W7 interface is defined through a three-dimensional {(1)H,(13)C}-edited-{(1)H,(12)C}-detected NOESY NMR experiment, and other aspects of the structure are modeled as perturbations to previously known coordinates and restraints.The structure supports the previously proposed troponin I blocking mechanism for the activity of W7 in striated muscle and suggests a role for the flexible tail of W7 in stabilization of the bound state.This clarifies the structure-activity relationships of W7 and implicates an electrostatically mediated component of activity in common analogues of W7, including the antipsychotic trifluoroperazine and the cardiotonic levosimendan.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

ABSTRACT
The calmodulin antagonist W7 binds to troponin C in the presence of Ca(2+) and inhibits striated muscle contraction. This study integrates multiple data into the structure of the regulatory domain of human cardiac troponin C (cNTnC) bound to Ca(2+) and W7. The protein-W7 interface is defined through a three-dimensional {(1)H,(13)C}-edited-{(1)H,(12)C}-detected NOESY NMR experiment, and other aspects of the structure are modeled as perturbations to previously known coordinates and restraints. The structure determination protocol optimizes the protein-W7 contacts prior to the introduction of protein-W7 steric interactions or conformational changes in the protein. The structure determination protocol gives families of conformers that all have an optimal docking as assessed by satisfaction of the target function. The structure supports the previously proposed troponin I blocking mechanism for the activity of W7 in striated muscle and suggests a role for the flexible tail of W7 in stabilization of the bound state. This clarifies the structure-activity relationships of W7 and implicates an electrostatically mediated component of activity in common analogues of W7, including the antipsychotic trifluoroperazine and the cardiotonic levosimendan.

Show MeSH
Related in: MedlinePlus